1. Field of the Invention
The present invention relates generally to biomolecules that mediate biological signal transduction in cells, which signals are communicated by phosphorylation and dephosphorylation of cellular proteins for processes such as cellular differentiation, activation, proliferation and survival. More specifically, the invention relates to specific interactions between the protein tyrosine phosphatase known as density enhanced phosphatase-1 (DEP-1) and several distinct cellular proteins, and to related compositions and methods.
2. Description of the Related Art
Protein tyrosine phosphorylation is an essential element in signal transduction pathways that control fundamental cellular processes including growth and differentiation, cell cycle progression, and cytoskeletal function. Briefly, the binding of hormones, cytokines, growth factors, or other ligands to a cognate receptor protein tyrosine kinase (PTK) triggers autophosphorylation of tyrosine residues in the receptor itself and phosphorylation of tyrosine residues in the enzyme's target substrates. Within the cell, tyrosine phosphorylation is a reversible process; the phosphorylation state of a particular tyrosine residue in a target substrate is governed by the coordinated action of both PTKs that catalyze phosphorylation and protein tyrosine phosphatases (PTPs) that catalyze dephosphorylation.
The PTPs are a large and diverse family of enzymes found ubiquitously in eukaryotes (Charbonneau and Tonks, Ann. Rev. Cell Biol. 8:463–93 (1993)). Structural diversity within the PTP family arises primarily from variation in non-catalytic (potentially regulatory) sequences that are linked to one or more highly conserved catalytic domains. In general, soluble cytoplasmic PTP forms are termed non-receptor PTPs and those with at least one non-catalytic region that traverses the cell membrane are termed receptor-like PTPs (RPTPs).
A variety of non-receptor PTPs have been identified that characteristically possess a single catalytic domain flanked by non-catalytic sequences. Such non-catalytic sequences have been shown to include, among others, sequences homologous to cytoskeletal-associated proteins (Yang et al., Proc. Natl. Acad. Sci. USA 88:5949–53 (1991)) or to lipid binding proteins (Gu et al., Proc. Natl. Acad. Sci. USA 89:2980–84 (1992)), and/or sequences that mediate association of the enzyme with specific intracellular membranes (Frangioni et al., Cell 68:545–60 (1992)), suggesting that subcellular localization may play a significant role in regulation of PTP activity.
Among RPTPs, analysis of non-catalytic domain sequences suggests their involvement in signal transduction mechanisms; however, binding of specific ligands to the extracellular segment of RPTPs has been characterized in only a few instances. For example, homophilic binding has been demonstrated between molecules of PTPμ (Brady-Kalnay et al., J. Cell. Biol. 122:961–972 (1993)) i.e., the ligand for PTPμ expressed on a cell surface is another PTPμ molecule on the surface of an adjacent cell. Little is otherwise known about ligands that specifically bind to, and modulate the activity of, the majority of RPTPs.
Many receptor-like PTPs comprise an intracellular carboxyl segment with two catalytic domains, a single transmembrane domain and an extracellular amino terminal segment (Krueger et al., EMBO J. 9:3241–52 (1990)). Subclasses of RPTPs are distinguished from one another on the basis of categories or “types” of extracellular domains (Fischer et al., Science 253:401–406 (1991)). Type I RPTPs have a large extracellular domain with multiple glycosylation sites and a conserved cysteine-rich region. CD45 is a typical Type I RPTP. The Type II RPTPs contain at least one amino terminal immunoglobulin (Ig)-like domain adjacent to multiple tandem fibronectin type III (FNIII)-like repeats. Similar repeated FNIII domains, believed to participate in protein-protein interactions, have been identified in receptors for IL2, IL4, IL6, GM-CSF, prolactin, erythropoietin, and growth hormone (Patthy, Cell 61:13–14 (1992)). The leukocyte common antigen-related PTP known as LAR exemplifies the Type II RPTP structure (Streuli et al., J. Exp. Med. 168:1523–30 (1988)), and, like other Type II RPTPs, contains an extracellular region reminiscent of the NCAM class of cellular adhesion molecules (Edelman and Crossin, Ann. Rev. Biochem. 60:155–190 (1991)). The Type III RPTPs, such as HPTPβ (Krueger et al., EMBO J. 9:3241–52 (1990)), contain only multiple tandem FNIII repeats in the extracellular domain. The Type IV RPTPs, for example RPTPα (Krueger et al. (1990) supra), have relatively short extracellular sequences lacking cysteine residues but containing multiple glycosylation sites. A fifth type of RPTP, exemplified by PTPγ (Barnes et al., Mol. Cell Biol. 13:1497–506 (1993)) and PTPζ (Krueger and Saito, Proc. Natl. Acad. Sci. USA 89:7417–21 (1992)), is characterized by an extracellular domain containing a 280 amino acid segment that is homologous to carbonic anhydrase (CAH) but lacks essential histidine residues required for reversible hydration of carbon dioxide.
Characteristics shared by both the soluble PTPs and the RPTPs include an absolute specificity for phosphotyrosine residues, a high affinity for substrate proteins, and a specific activity that is one to three orders of magnitude in excess of that of the PTKs in vitro (Fischer et al., Science 253:401–406 (1991); Tonks, Curr. Opin. Cell. Biol. 2:1114–24 (1990)). Supporting a significant physiological role for PTP activity is the observation that treatment of NRK-1 cells with vanadate, a potent inhibitor of PTP activity, resulted in enhanced levels of phosphotyrosine and generation of a transformed cellular morphology (Klarlund, Cell 41:707–17 (1985)). This observation implies potential therapeutic value for PTPs and agents that modulate PTP activity as indirect modifiers of PTK activity and, thus, levels of cellular phosphotyrosine.
Other studies have also highlighted aspects of the physiological importance of PTP activity. For example, mutations in the gene encoding a non-receptor hematopoietic cell protein tyrosine phosphatase, HCP, have been shown to result in severe immune dysfunction characteristic of the motheaten phenotype in mice (Schultz et al., Cell 73:1445–54 (1993)). Under normal conditions HCP may act as a suppressor of PTK-induced signaling pathways, for example, the CSF-1 receptor (Schultz et al., supra). Some PTP enzymes may be the products of tumor suppressor genes, and their mutation or deletion may contribute to the elevation in cellular phosphotyrosine associated with certain neoplasias (Brown-Shimer et al., Cancer Res. 52:478–82 (1992); Wary et al., Cancer Res. 53:1498–502 (1993)). Mutations observed in the gene for RPTPγ in murine L cells would be consistent with this hypothesis (Wary et al., Cancer Res. 53:1498–502 (1993)). The observation that the receptor-like PTP CD45 is required for normal T cell receptor-induced signaling (Pingel et al., Cell 58:1055–65 (1989)) provides evidence implicating PTP activity as a positive mediator of cellular signaling responses. Mice homozygous for a disrupted PTP-1B gene (PTP-1B −/−) exhibited enhanced sensitivity to insulin and resistance to weight gain, relative to controls having functional PTP-1B (Elchebly et al., 1999 Science 283:1544).
A variety of ligands trigger the reversible phosphorylation of tyrosyl residues in cellular proteins, a process that underlies the control of such fundamental cellular functions as growth and proliferation, migration and morphogenesis. Tyrosine phosphorylation is regulated by the coordinated action of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Classically it was thought that the PTKs provided the “on-switch” to initiate a physiological response, whereas the PTPs functioned to counteract the PTKs and to return the system to its basal state. However, it has been shown that PTPs may themselves function positively to promote signaling, for example by promoting the dephosphorylation and activation of PTKs, thus coordinating with, rather than antagonizing PTK function (reviewed in (Hermiston et al., J. Clin. Invest. 109:9–14 (2002)). A further level of complexity has been introduced with the realization that whether a defined PTP functions positively or negatively may depend upon the signaling context. Thus, SHP-2 is an activator of signaling through the HGF/SF receptor Met (Maroun et al., Mol. Cell Biol. 20:8513–25 (2000)) and the EGF receptor (Bennett et al., Mol. Cell Biol. 16:1189–202 (1996)), but is an inhibitor of signaling through the PDGF receptor (Meng et al., Mol. Cell 9:387–99 (2002)). Following ligand binding, a receptor PTK may become phosphorylated on multiple tyrosine residues, which serve as docking sites for distinct signaling proteins. The spectrum of such signaling molecules that associate with the PTK will determine the nature of the response that is initiated following ligand stimulation. The possibility exists, therefore, that a PTP may dephosphorylate a particular site in a receptor PTK and thereby determine the signaling outcome of a particular stimulus. Thus, dephosphorylation of receptor PTKs by members of the PTP family may function as a mechanism for regulating the specificity of a signaling event rather than simply as an “off-switch.”
Normal cells in culture exhibit contact inhibition of growth, that is, as adjacent cells in a confluent monolayer touch each other, their growth is inhibited (Stoker et al., Nature 215:171–72 (1967)). Because PTKs promote cell growth, PTP action may underlie mechanisms of growth inhibition. Density Enhanced PTP-1 (DEP-1) is a Type III receptor PTP whose expression is enhanced as cells approach confluence (Ostman et al., Proc. Natl. Acad. Sci. USA 91:9680–84 (1994)). Initially cloned from human cDNA libraries (U.S. Pat. No. 6,114,140; WO95/30008), DEP-1 homologues were subsequently identified in rat and mouse (Kuramochi et al., FEBS Lett. 378:7–14 (1996); Borges et al., Circ. Res. 79:570–80 (1996)).
DEP-1 comprises an extracellular segment of eight-fibronectin type III repeats, a transmembrane domain and a single cytoplasmic PTP domain. Also known as PTP-η (Honda et al., Blood 84:4186–94 (1994)) and CD 148 (Palou et al., Immunol. Lett. 57:101–103 (1997)), DEP-1 is expressed in a variety of tissues and cell types. There is a growing body of evidence suggesting a role for DEP-1 in the inhibition of cell growth. After vascular injury DEP-1 expression is down regulated in migrating and proliferating rat endothelial cells (Borges et al., supra). Attempts have been made to express DEP-1 constitutively in breast cells and macrophages (Keane et al., Cancer Res. 56:4236–43 (1996); Osborne et al., J. Leukoc. Biol. 64:692–701 (1998)), however, this inhibited development of stable cell lines, further reinforcing a role for DEP-1 in growth inhibition.
In addition to its role in growth inhibition, DEP-1 has also been implicated in differentiation. The levels of DEP-1 mRNA are increased in various cell lines in response to factors that lead to differentiation (Borges et al., supra; Keane et al., supra; Zhang et al., Exp. Cell Res. 235:62–70 (1997); Martelli et al., Exp. Cell Res. 245:195–202 (1998)). Interestingly, in rat thyroid cells the expression of DEP-1 (rPTP-TI) mRNA decreases with increasing levels of transformation (Zhang et al., supra; Florio et al., Endocrinology 138:3756–63 (1997)). Re-introduction of DEP-1 into the transformed cells leads to reduced growth rates, stabilization of the cyclin-dependent kinase inhibitor p27kip1 and partial re-acquisition of a differentiated phenotype (Trapasso et al., Mol. Cell Biol. 20:9236–46 (2000)). Loss of DEP-1 expression has also been observed in human thyroid tumors (id.). Furthermore, the DEP-1 gene Ptprj was identified as a positional candidate for the mouse colon-cancer susceptibility locus Scc1(Ruivenkamp et al., Nat. Genet. 31:295–300 (2002)). Frequent deletions, loss of heterozygosity (LOH) and missense mutations in the human Ptprj gene have also been identified in colon, lung and breast cancers (id.). Taken together these data indicate that DEP-1 may be a critical factor in controlling cellular growth and transformation.
DEP-1 has recently been shown to localize at cell borders in endothelial cells and its staining pattern overlapped with that of the functional protein VE-cadherin (Takahashi et al., J. Am. Soc. Nephrol. 10:2135–45 (1999)). Interestingly, members of the cadherin family of cell-cell adhesion molecules function in the suppression of cell growth and tumor invasion. Junctional components such as β-catenin, however, can also promote cell growth by inducing the transcription of genes involved in proliferation and cancer progression (reviewed in Ben-Ze'ev et al., Exp. Cell Res. 261:75–83 (2000)). The growth inhibitory effects of cadherins may involve binding and sequestration of the signaling pool of the catenins (Gottardi et al., J. Cell Biol. 153:1049–60 (2001); Stockinger et al., J. Cell Biol. 154:1185–96 (2001)). Reversible tyrosine phosphorylation is an important aspect of the regulation of functional integrity and the control of signals emanating from these sites (reviewed in Conacci-Sorrell et al., J. Clin. Invest. 109:987–91 (2002)).
Clearly there is a need for the identification of PTPs, PTKs and other components of biological signal transduction pathways that interact with members of these enzyme families, in order to better understand the cellular and molecular mechanisms that govern such processes as cell growth, differentiation and survival in normal and pathological conditions. For instance, determination of the PTKs and PTPs that act upon the components of cell junctions will be important for understanding the regulation of cell morphology and the control of gene expression, events that ultimately influence growth and migration. The present invention contributes to such understanding of the biological signal transduction pathways in which DEP-1 functions by identifying several proteins with which DEP-1 specifically interacts, and offers other related advantages.